Cryptosporidiosis is a globally important disease which is underappreciated as a cause of morbidity and mortality in children. It is also common in persons with AIDS and in children with persistent diarrhea who suffer growth retardation and malnutrition. No effective therapy exists. The recent Global Enteric Multicenter Study (GEMS) reported that moderate-to-severe diarrheal disease in the pediatric population in 4 sites in sub-Saharan Africa and 3 sites in South Asia, Cryptosporidium was second only to rotavirus as a cause of disease in children < 2 years of age. This application proposes to develop Cryptosporidium vaccines for humans. There are two species of this enteric parasite, C. hominis and C. parvum, both of which cause illness in humans, although C. hominis is more pathogenic and more frequently associated with disease in humans. The gnotobiotic (GB) piglet is the only model that results in diarrhea following challenge with C. hominis, while C. parvum infects all mammals including humans. Piglets that recover from Cryptosporidium diarrhea are significantly protected when re-challenged with the homologous species (Aim 1), and partially protected against C. parvum. We propose to perform a series of studies in piglets to address the fundamental hypothesis that specific antibodies protect against Cryptosporidium. Specific Aim 1: Re-demonstrate that C. hominis or C. parvum diarrheal infection of GB piglets results in active infection-derived immunity that is protective against parasite rechallenge. Specific Aim 2: Determine whether passive transfer of antibodies from immunized sows confers passive protection to their suckling offsprings against challenge with the homologous species. Specific Aim 3: Determine whether passive transfer of circulating or intestinal antibodies from immunized sows can protect GB piglets challenged with homologous or heterologous species. Specific Aim 4: Identify surface antigens of C. hominis and C. parvum sporozoites using immunoproteomics and reverse vaccinology approaches. Serum IgG and milk IgA from Aims 1 and 2 will be used in immunoproteomic techniques to identify antigen targets of these antibodies. Specific Aim 5: Determine whether putative C. hominis and C. parvum protective antigens identified in Aims 4 & 5 will protect GB piglets or mice when administered a) parenterally as purified proteins, b) expressed by a Salmonella Typhi live vector or c) in a prime boost strategy (prime with live vector followed by a boost with purified antigen).